This invention relates to a new use of a compound possessing an inhibitory activity on the production of nitric oxide, which is useful in a medical field.
Various compounds possessing an inhibitory activity on the production of nitric oxide have already been known, for example, in EP 0 394 989-A2, WO96/16981, WO97/45425, WO98/27108, etc.
This invention relates to a new use of a compound possessing an inhibitory activity on the production of nitric oxide, for increasing an effect caused by interleukin 2 inhibitor (hereinafter, referred to IL-2 inhibitor).
Therefore, one object of the present invention is to provide a new use of a compound possessing an inhibitory activity on the production of nitric oxide, for increasing an effect caused by IL-2 inhibitor.
Another object of this invention is to provide a method for increasing an effect caused by IL-2 inhibitor by administering an effective amount of a compound possessing an inhibitory activity on the production of nitric oxide.
A further object of this invention is to provide a use of a compound possessing an inhibitory activity on the production of nitric oxide for manufacturing a medicament for increasing an effect caused by IL-2 inhibitor.
Still further object of this invention is to provide a composition comprising a compound possessing an inhibitory activity on the production of nitric oxide, for increasing an effect caused by IL-2 inhibitor.
In the present invention, the xe2x80x9ccompound possessing an inhibitory activity on the production of nitric oxidexe2x80x9d should not be limited and be considered to mean any compounds which have an inhibitory activity on the production of nitric oxide. Preferable one is a compound possessing an inhibitory activity on the production of inducible nitric oxide synthase (iNOS), and the other preferable one is a compound possessing an iNOS-inhibitory activity.
The compound having the following formula (I) are exemplified as a preferable example of the above xe2x80x9ccompound possessing an inhibitory activity on the production of nitric oxidexe2x80x9d. 
wherein
R1 is indolyl which may have a suitable substituent selected from the group consisting of lower alkyl, phenyl, halogen, lower alkoxy, and nitro, benzofuranyl, phenyl which may have one or two suitable substituent(s) selected from the group consisting of amino, acylamino, lower alkylamino, halogen, lower alkoxy and nitro, lower alkyl, quinoxalinyl, quinolyl, pyrrolyl, pyrimidinyl having benzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, benzoxazolyl, indolinyl, anilino, phenylcarbamoyl or imidazolyl which may have one or two suitable substituent(s) selected from the group consisting of phenyl, lower alkyl and indolyl;
R2 is hydrogen or phenyl(lower)alkyl;
R4 is hydrogen, phenyl or pyridyl, each of which may have suitable substituent(s) selected from the group consisting of lower alkyl, lower alkoxy, lower alkylthio, halogen, trihalomethyl, nitro, cyano, imidazolyl, optionally protected hydroxy, acyl, amino, acylamino, diacylamino, di(lower)alkyl-amino, amino(lower)alkyl, acylamino(lower)alkyl, pyrazolyl, morpholinyl, piperidyl, triazolyl, lower alkoxy(lower)alkoxy, hydroxy(lower)alkyl, lower alkylpiperazinyl, phenyl and carboxy, quinolyl or 3,4-methylenedioxyphenyl;
R5 is hydrogen, imidazolyl, phenyl, nitrophenyl, phenyl(lower)- alkyl, optionally esterified carboxy or a group of the formula: 
in which R7 and R8 are the same or different and each is hydrogen, phenyl, phenyl(lower)alkyl, lower alkyl or lower alkoxy; or
R4 and R5 in combination form a group of the formula:
xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94
Y is a group of the formula: 
in which R3 is hydrogen or a group of the formula:
xe2x80x83xe2x80x94(CH2)nxe2x80x94R6
in which R6 is optionally protected hydroxy, acyl, carboxy, acylamino, lower alkoxy, phenyl(lower)alkoxy, lower alkylthio, phenyl which may have a suitable substituent selected from the group consisting of lower alkoxy, halogen, amino, acylamino, diacylamino and nitro, pyridyl which may have a suitable substituent selected from the group consisting of lower alkoxy and halogen, pyrazinyl, pyrimidinyl, furyl, imidazolyl, naphthyl, N-(lower)alkylindolyl or 3,4-methylenedioxyphenyl, and n is an integer of 0 to 3,
or a group of the formula: 
in which R11 is phenyl, phenoxy or phenyl(lower)alkoxy; or
R2 and R3 in combination form a group of the formula: 
m is O or 1; and
X is S or NR9 
in which R9 is hydrogen, lower alkyl, cyclo(lower)alkyl or a group of the formula: 
in which R10 is hydrogen, lower alkyl or lower alkoxy;
or a pharmaceutically acceptable salt thereof.
Suitable pharmaceutically acceptable salts of the compound (I) are conventional non-toxic salts and include, for example, a salt with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt (e.g., sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt; a salt with an organic base, for example, an organic amine salt (e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,Nxe2x80x2-dibenzylethylenediamine salt, etc.); an inorganic acid addition salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.); an organic carboxylic or sulfonic acid addition salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, citrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.); and a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid,gultamic acid, etc.).
In the above and subsequent descriptions of the present specification, suitable examples and illustration of the various definitions which the present invention intends to include within the scope thereof are explained in detail as follows.
The term xe2x80x9clowerxe2x80x9d is used to intend a group having 1 to 6, preferably 1 to 4, carbon atom(s), unless otherwise provided.
Suitable xe2x80x9clower alkylxe2x80x9d and xe2x80x9clower alkyl moietyxe2x80x9d in the terms xe2x80x9clower alkylthioxe2x80x9d, xe2x80x9clower alkylthio(lower)alkylxe2x80x9d, xe2x80x9cN-(lower)alkylindolylxe2x80x9d, xe2x80x9clower alkylaminoxe2x80x9d, xe2x80x9cdi(lower)alkylaminoxe2x80x9d, xe2x80x9cphenyl(lower)alkylxe2x80x9d, xe2x80x9camino(lower)alkylxe2x80x9d, xe2x80x9cacylamino(lower)alkylxe2x80x9d, xe2x80x9chydroxy(lower)alkylxe2x80x9d and xe2x80x9clower alkylpiperazinylxe2x80x9d include straight or branched onehaving 1 to 6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl andhexyl, in which more preferred one is C1-C4 alkyl.
Suitable xe2x80x9clower alkoxyxe2x80x9d and xe2x80x9clower alkoxy moietyxe2x80x9d in the terms xe2x80x9clower alkoxy(lower)alkoxyxe2x80x9d and xe2x80x9cphenyl(lower)alkoxyxe2x80x9d include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, tert-pentyloxy and hexyloxy, in which more preferred one is C1-C4 alkoxy.
Suitable xe2x80x9chalogenxe2x80x9d includes, for example, fluorine, bromine, chlorine and iodine.
xe2x80x9cOptionally esterified carboxyxe2x80x9d includes carboxy and esterified carboxy. Suitable examples of said ester include lower alkyl ester(e.g., methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, tert-butyl ester, pentyl ester, tert-pentyl ester, hexyl ester, etc.); lower alkenyl ester (e.g., vinyl ester, allylester, etc.); lower alkynyl ester (e.g., ethynyl ester, propynyl ester, etc.); lower alkoxy(lower)alkyl ester (e.g., methoxymethyl ester, ethoxymethyl ester, isopropoxymethyl ester, 1-methoxyethyl ester, 1-ethoxyethyl ester, etc.); mono(or di or tri)aryl(lower)alkyl ester, for example, mono(or di or tri)phenyl(lower)alkyl ester which may have one or more suitable substituent(s) [e.g.,benzyl ester, 4-methoxybenzyl ester, 4-nitrobenzyl ester, phenethyl ester, trityl ester, benzhydryl ester, bis(methoxyphenyl)methyl ester, 3,4-dimethoxybenzyl ester, 4-hydroxy-3,5-di-tert-butylbenzyl ester, etc.]; and aryl ester which may have one or more suitable substituent(s)such as substituted or unsubstituted phenyl ester (e.g., phenyl ester, tolyl ester, tert-butylphenyl ester, xylyl ester, mesityl ester, cumenyl ester, 4-chlorophenyl ester, 4-methoxyphenyl ester, etc.).
Suitable xe2x80x9ctrihalomethylxe2x80x9d includes, for example, trifluoromethyl,trichloromethyl and tribromomethyl, in which preferred one is trifluoromethyl.
Suitable xe2x80x9camino protective groupxe2x80x9d includes, for example, acyl and conventional protective group such as mono(or di or tri)aryl(lower)-alkyl, for example, mono(or di or tri)phenyl(lower)alkyl (e.g., benzyl, trityl, etc.).
Suitable xe2x80x9cacylxe2x80x9d and xe2x80x9cacyl moietyxe2x80x9d in the terms xe2x80x9cacylaminoxe2x80x9d, xe2x80x9cdiacylaminoxe2x80x9d and xe2x80x9cacylamino(lower)alkylxe2x80x9d include, for example, carbamoyl which may be substituted by suitable substituent(s), aliphatic acyl group and acyl group containing an aromatic ring, which is referred to as aromatic acyl, or a heterocyclic ring, which is referred to as heterocyclic acyl.
Suitable examples of said acyl are illustrated as follows: xe2x80x9ccarbamoyl which may be substituted by suitable substituent(s)xe2x80x9d includes a group of the formula: 
wherein R12 and R13 are the same or different and each is hydrogen, lower alkyl, phenyl which may have a suitable substituent selected from the group consisting of lower alkoxy and halogen, phenyl (lower) alkyl, pyridyl, pyridyl (lower)alkyl or 3,4-methylenedioxyphenyl;
aliphatic acyl such as lower alkanoyl which may be substituted by oneto three halogen atoms (e.g., formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, trichloroacetyl, trifluoroacetyl, etc.), lower alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, tert-pentyloxycarbonyl, etc.), lower alkylsulfonyl (e.g., methylsulfonyl, ethylsulfonyl, etc.), lower alkoxysulfonyl (e.g., methoxysulfonyl, ethoxysulfonyl, etc.), cyclo(lower)alkylcarbonyl (e.g., cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), and the like;
aromatic acyl such as aroyl (e.g., benzoyl, toluoyl, naphthoyl, etc.), aryl(lower)alkanoyl [e.g., phenyl(lower)alkanoyl (e.g., phenylacetyl, phenylpropanoyl, phenylbutanoyl, etc.), naphthyl(lower)alkanoyl (e.g., naphthylacetyl, naphthylpropanoyl, naphthylbutanoyl, etc.), etc.], aryl(lower)alkoxycarbonyl [e.g., phenyl(lower)alkoxycarbonyl (e.g., benzyloxycarbonyl, etc.), etc.], aryloxycarbonyl (e.g., phenoxycarbonyl, naphthyloxycarbonyl, etc.), aryloxy(lower)alkanoyl (e.g., phenoxyacetyl, phenoxypropionyl, etc.), arylsulfonyl (e.g., phenylsulfonyl, p-tolylsufonyl, etc.), and the like;
heterocyclic acyl such as indolylcarbonyl (e.g., indolyl-2-yl-carbonyl, etc.), benzofuranylcarbonyl (e.g., benzofuran-2-yl-carbonyl), quinoxalinylcarbonyl, quinolylcarbonyl, pyrrolylcarbonyl, benzimidazolylcarbonyl, benzothienylcarbonyl, benzo-thiazolylcarbonyl, imidazolylcarbonyl,pyridylcarbonyl, morpholinylcarbonyl (e.g., morpholinocarbonyl) and the like. Optionally protected hydroxyxe2x80x9d includes hydroxy and protected hydroxy. Suitable examples of xe2x80x9chydroxy protective groupxe2x80x9d in the term xe2x80x9cprotected hydroxyxe2x80x9d include acyl (e.g., acetyl, trichloroacetyl, etc.), mono(or di or tri)phenyl(lower)alkyl which may have one or more suitable substituent(s) (e.g., benzyl, 4-methoxybenzyl, trityl, etc.), trisubstituted silyl [e.g., tri(lower)alkylsilyl (e.g., trimethylsilyl, tert-butyl-dimethylsilyl, etc.), etc.], tetrahydropyranyl and the like.
Suitable xe2x80x9cprotected carboxyxe2x80x9d is carboxy group protected by conventional protective group such as lower alkoxycarbonyl [e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, sec-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, neopentyloxycarbonyl, hexyloxycarbonyl, etc.], optionally substituted phenyl (lower)alkoxycarbonyl for exemple, mono- or di- or tri-phenyl(lower)alkoxycarbonyl which may be substituted by nitro [e.g., benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, benzhydryloxycarbonyl, trityloxycarbonyl, etc.] and the like.
Suitable xe2x80x9ccyclo(lower)alkylxe2x80x9d includes cycloalkyl having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, in which more preferred ones are cyclopropyl and cyclobutyl.
The term xe2x80x9cmorpholinylxe2x80x9d includes 2-morpholinyl, 3-morpholinyl and 4-morpholinyl (i.e. morpholino).
The term piperidylxe2x80x9d includes 1-piperidyl (i.e. piperidino), 2-piperidyl, 3-piperidyl and 4-piperidyl.
And further, the compound having the following formula (II) is also exemplified as the preferable one of the xe2x80x9ccompound possessing an inhibitory activity on the production of nitric oxidexe2x80x9d. 
wherein,
R21 is benzofuranyl having halogen,
R22 is lower alkyl, and
R23 is morpholinyl.
The compound (I) and (II) usable in the present invention can be prepared in a similar manner to that of WO98/27108 (PCT/JP97/04243), the disclosure of which is incorporated herein by reference.
It is to be noted that the compound (I), (II) and other compounds may include one or more stereoisomer(s) such as optical isomer(s) and geometrical isomer(s) due to asymmetric carbon atom(s) and double bond(s), and all of such isomers and mixtures thereof are included within the scope of this invention.
The compound (I), (II) and pharmaceutically acceptable salts thereof can be in a form of solvates [e.g., hydrate, ethanolate, etc.], which are included within the scope of the present invention.
And further, the compound (I), (II) and pharmaceutically acceptable salts thereof can be in a form of pro-drugs, suitable derivatives, and so on.
The compound (I), (II) and pharmaceutically acceptable salts thereof possesses a strong inhibitory activity on the production of nitric oxide (NO). They are expected to possess a nitric oxide synthase (NOS)-inhibitory activity or a NOS-production inhibitory activity.
And further, the compounds shown in EP 0 394 989-A2, WO 96/16981, WO 97/45425, the Japanese Patent publication No. 10-45751, the Japanese patent application No. 9-160128, and so on, can also be used as xe2x80x9cthe compound possessing the inhibitory activity on the production of nitric oxidexe2x80x9d.
The xe2x80x9cIL-2 inhibitorxe2x80x9d used in the present invention should not be limited and be considered to mean any ones possessing IL-2 inhibitory activity. The particular example is the one possessing an inhibitory activity on the production of IL-2. And the other is the one that inhibits the transmission of IL-2 signal.
IL-2 is known to mediate immune system. Therefore, the preferable xe2x80x9ceffect caused by interleukin 2 inhibitorxe2x80x9d is an immunosuppressive activity. Particularly, xe2x80x9cthe effect caused by interleukin 2 inhibitorxe2x80x9d may be the treatment and prevention of rejection by transplantation, Graft-versus-Host diseases by medulla ossium transplantation, autoimmune diseases, and so on. And the present invention is useful to suppress immune reaction, to prolong the survival period of the graft, to reduce the administration amount of IL-2 inhibitor, and/or to reduce undesirable side effects caused by IL-2 inhibitor.
Preferable xe2x80x9cIL-2 inhibitorxe2x80x9d is, for example, the tricyclic macrolide shown in EP-0184162, WO89/05303, WO93/05058, WO96/31514, and so on, the disclosure of which is incorporated herein by reference. It is well known that those tricyclic macrolides have strong IL-2 inhibitory activity.
As a particular example of the tricyclic macrolides compounds, the tricyclic compound of the following formula (III) can be exemplified. 
(wherein each of adjacent pairs of R1 and R2, R3 and R4, and R5 and R6 independently
(a) is two adjacent hydrogen atoms, but R2 may also be an alkyl group or
(b) may form another bond formed between the carbon atoms to which they are attached;
R7 is a hydrogen atom, a hydroxy group, a protected hydroxy group, or an alkoxy group, or an oxo group together with R1;
R8 and R9 are independently a hydrogen atom or a hydroxy group;
R10 is a hydrogen atom, an alkyl group, an alkyl group substituted by one or more hydroxy groups, an alkenyl group, an alkenyl group substituted by one or more hydroxy groups, or an alkyl group substituted by an oxo group;
X is an oxo group, (a hydrogen atom and a hydroxy group), (a hydrogen atom and a hydrogen atom), or a group represented by the formula xe2x80x94CH2Oxe2x80x94;
Y is an oxo group, (a hydrogen atom and a hydroxy group), (a hydrogen atom and a hydrogen atom), or a group represented by the formula Nxe2x80x94NR11R12 or Nxe2x80x94OR13;
R11 and R12 are independently a hydrogen atom, an alkyl group, an aryl group or a tosyl group;
R13, R14, R15, R16, R17, R18, R19, R22 and R23 are independently a hydrogen atom or an alkyl group;
R24 is an optionally substituted ring system which may contain one or more heteroatoms;
n is an integer of 1 or 2; and
in addition to the above definitions, Y, R10 and R23, together with the carbon atoms to which they are attached, may represent a saturated or unsaturated 5- or 6-membered nitrogen, sulfur and/or oxygen containing heterocyclic ring optionally substituted by one or more groups selected from the group consisting of an alkyl, a hydroxy, an alkoxy, a benzyl, a group of the formula xe2x80x94CH2Se(C6H5), and an alkyl substituted by one or more hydroxy groups.
Preferable R24 may be cyclo (C5-7) alkyl group, and the following ones can be exemplified.
(a) a 3,4-di-oxo-cyclohexyl group;
(b) a 3-R20-4-R21-cyclohexyl group,
in which R20 is hydroxy, an alkoxy group, an oxo group, or a xe2x80x94OCH2OCH2CH2OCH3 group, and
R21 is hydroxy, xe2x80x94OCN, an alkoxy group, a heteroaryloxy which may be substituted by suitable substituents, a xe2x80x94OCH2OCH2CH2OCH3 group, a protected hydroxy group, chloro, bromo, iodo, aminooxalyloxy, an azido group, p-tolyloxythiocarbonyloxy, or R25R26CHCOOxe2x80x94,
in which R25 is optionally protected hydroxy or protected amino, and R26 is hydrogen or methyl, or
R20 and R21 together form an oxygen atom in an epoxide ring; or
(c) cyclopentyl group substituted by methoxymethyl, optionally protected hydroxymethyl, acyloxymethyl (in which the acyl moiety optionally contains either a dimethylamino group which may be quaternized, or a carboxy group which may be esterified), one or more amino and/or hydroxy groups which may be protected, or aminooxalyloxymethyl. A preferred example is a 2-formyl-cyclopentyl group.
The definitions used in the above general formula (III) and the specific and preferred examples thereof are now explained and set forth in detail.
The term xe2x80x9clowerxe2x80x9d means, unless otherwise indicated, a group having 1 to 6 carbon atoms.
Preferable examples of the xe2x80x9calkyl groupsxe2x80x9d and an alkyl moiety of the xe2x80x9calkoxy groupxe2x80x9d include a straight or branched chain aliphatic hydrocarbon residue, for example, a lower alkyl group such asmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl and hexyl.
Preferable examples of the xe2x80x9calkenyl groupsxe2x80x9d include a straight or branched chain aliphatic hydrocarbon residue having one double-bond, for example, a lower alkenyl group such as vinyl, propenyl (e.g., allyl group), butenyl, methylpropenyl, pentenyl and hexenyl.
Preferable examples of the xe2x80x9caryl groupsxe2x80x9d include phenyl, tolyl, xylyl, cumenyl, mesityl and naphthyl.
Preferable protective groups in the xe2x80x9cprotected hydroxy groupsxe2x80x9d and the xe2x80x9cprotected aminoxe2x80x9d are 1-(lower alkylthio)(lower)alkyl group such as a lower alkylthiomethyl group (e.g., methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, hexylthiomethyl, etc.), more preferably C1-C4 alkylthiomethyl group, most preferably methylthiomethyl group;
trisubstituted silyl group such as a tri(lower)alkylsilyl (e.g., trimethylsilyl, triethylsilyl, tributylsilyl, tert-butyldimethylsilyl, tri-tert-butylsilyl, etc.) or lower alkyl-diarylsilyl (e.g., methyldiphenylsilyl, ethyldiphenylsilyl, propyldiphenylsilyl, tert-butyldiphenylsilyl, etc.), more preferably tri(C1-C4)alkylsilyl group and C1-C4 alkyldiphenylsilyl group, most preferably tert-butyldimethylsilyl group and tert-butyldiphenylsilyl group; and an acyl group such as an aliphatic, aromatic acyl group or an aliphatic acyl group substituted by an aromatic group, which are derived from a carboxylic acid, sulfonic acid or carbamic acid.
Examples of the aliphatic acyl groups include a lower alkanoyl group optionally having one or more suitable substituents such as carboxy, e.g., formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, carboxyacetyl, carboxypropionyl, carboxybutyryl, carboxyhexanoyl, etc.; a cyclo(lower)alkoxy(lower)alkanoyl group optionally having one or more suitable substituents such as lower alkyl, e.g., cyclopropyloxyacetyl, cyclobutyloxypropionyl, cycloheptyloxybutyryl, menthyloxyacetyl, menthyloxypropionyl, menthyloxybutyryl, menthyloxypentanoyl, menthyloxyhexanoyl, etc.; a camphorsulfonyl group; or a lower alkylcarbamoyl group having one or more suitable substituents such as carboxy or protected carboxy, for example, carboxy(lower)alkylcarbamoyl group (e.g., carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl, etc.), tri(lower)alkylsilyl(lower)alkoxycarbonyl(lower)alkylcarbamoyl group (e.g., trimethylsilylmethoxycarbonylethylcarbamoyl, trimethylsilylethoxycarbonylpropylcarbamoyl, triethylsilylethoxycarbonylpropylcarbamoyl, tert-butyldimethylsilylethoxycarbonylpropylcarbamoyl, tri-methylsilylpropoxycarbonylbutylcarbamoyl, etc.) and so on.
Examples of the aromatic acyl groups include an aroyl group optionally having one or more suitable substituents such as nitro, e.g., benzoyl, toluoyl, xyloyl, naphthoyl, nitrobenzoyl, dinitrobenzoyl, nitronaphthoyl, etc.; and an arenesulfonyl group optionally having one or more suitable substituents such as halogen, e.g., benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, iodobenzenesulfonyl, etc.
Examples of the aliphatic acyl groups substituted by an aromatic group include a (lower)alkanoyl group optionally having one or more suitable substituents such as lower alkoxy or trihalo(lower)alkyl, e.g., phenylacetyl, phenylpropionyl, phenylbutyryl, 2-trifluoromethyl-2-methoxy-2-phenylacetyl, 2-ethyl-2-trifluoromethyl-2-phenylacetyl, 2-trifluoromethyl-2-propoxy-2-phenylacetyl, etc.
More preferable acyl groups among the aforesaid acyl groups are C1-C4 alkanoyl group optionally having carboxy, cyclo(C5-C6)alkoxy(C1-C4)alkanoyl group having two (C1-C4) alkyls at the cycloalkyl moiety, camphorsulfonyl group, carboxy(C1-C4)alkylcarbamoyl group, tri(C1-C4)alkylsilyl(C1-C4)alkoxycarbonyl(C1-C4)alkylcarbamoyl group, benzoyl group optionally having one or two nitro groups, benzenesulfonyl group having halogen, or phenyl(C1-C4)alkanoyl group having C1-C4 alkoxy and trihalo(C1-C4)alkyl group. Among these, the most preferable ones are acetyl, carboxypropionyl, menthyloxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and 2-trifluoromethyl-2-methoxy-2-phenylacetyl.
Preferable examples of the xe2x80x9c5- or 6-membered nitrogen, sulfur and/or oxygen containing heterocyclic ringxe2x80x9d include a pyrrolyl group and a tetrahydrofuryl group.
The tricyclic compounds (III) and its pharmaceutically acceptable salt for use in accordance with this invention are well known to have excellent immunosuppressive activity, antimicrobial activity and other pharmacological activities and, as such, be of value for the treatment or prevention of rejection reactions by transplantation of organs or tissues, graft-vs-host diseases, autoimmune diseases, and infectious diseases [EP-A-0184162, EP-A-0323042, EP-A-423714, EP-A-427680, EP-A-465426, EP-A-480623, EP-A-532088, EP-A-532089, EP-A-569337, EP-A-626385, WO89/05303, WO93/05058, WO96/31514, WO91/13889, WO91/19495, WO93/5059, etc.].
Particularly, the compounds which are designated as FR900506 (=FK506), FR900520 (ascomycin), FR900523, and FR900525 are products produced by microorganisms of the genus Streptomyces, such as Streptomyces tsukubaensis No. 9993 [deposited with National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology (formerly Fermentation Research Institute Agency of Industrial Science and Technology ), at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, date of deposit Oct. 5, 1984, accession number FERM BP-927] or Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 [deposited with National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology (formerly Fermentation Research Institute Agency of Industrial Science and Technology ), at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, date of deposit Jan. 12, 1985, accession number FERM BP-928] [EP-A-0184162]. The FK506 (general name: tacrolimus) of the following chemical formula, in particular, is a representative compound. 
Chemical name: 17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]octacos-18-ene-2,3,10,16-tetraone
The preferred examples of the tricyclic compounds (III) are the ones, wherein each of adjacent pairs of R3 and R4 or R5 and R6 independently form another bond formed between the carbon atoms to which they are attached;
each of R8 and R23 is independently a hydrogen atom;
R9 is a hydroxy group;
R10 is a methyl group, an ethyl group, a propyl group or an allyl group;
X is (a hydrogen atom and a hydrogen atom) or an oxo group;
Y is an oxo group;
each of R14, R15, R16, R17, R18, R19, and R22 is a methyl group;
R24 is a 3-R20-4-R21-cyclohexyl group,
in which R20 is hydroxy, an alkoxy group, an oxo group, or a xe2x80x94OCH2OCH2CH2OCH3 group, and
R21 is hydroxy, xe2x80x94OCN, an alkoxy group, a heteroaryloxy which may be substituted by suitable substituents, a xe2x80x94OCH2OCH2CH2OCH3 group, a protected hydroxy group, chloro, bromo, iodo, aminooxalyloxy, an azido group, p-tolyloxythiocarbonyloxy, or R25R26CHCOOxe2x80x94, in which R25 is optionally protected hydroxy or protected amino, and
R26 is hydrogen or methyl, or
R20 and R21 together form an oxygen atom in an epoxide ring; and
n is an integer of 1 or 2.
The most preferable tricyclic compounds (III) is, in addition to FK506, ascomycin derivatives such as halogenated-ascomycin (e.g., 33-epi-chloro-33-desoxyascomycin), which is disclosed in EP 427,680, example 66a.
As the other preferable example of the IL-2 inhibitor, rapamycin [THE MERCK INDEX (12th edition), No. 8288] and its derivatives can be exemplified. Preferred example of the derivatives is an O-substituted derivative in which the hydroxy in position 40 of formula A illustrated at page 1 of WO 95/16691, incorporated herein by reference, is replaced by xe2x80x94OR1 in which R1 is hydroxyalkyl, hydroalkoxyalkyl, acyiaminoalkyl and aminoalkyl; for example 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin and 40-O-(2-acetaminoethyl)rapamycin. These O-substituted derivatives may be produced by reacting rapamycin (or dihydro or deoxo-rapamycin) with an organic radical attached to a leaving group (for example RX where R is the organic radical which is desired as the O-substituent, such as an alkyl, allyl, or benzyl moiety, and X is a leaving group such as CCl3C(NH)O or CF3SO3) under suitable reaction conditions. The conditions may be acidic or neutral conditions, for example in the presence of an acid like trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their respective pyridinium or substituted pyridinium salts when X is CCl3C(NH)O or in the presence of a base like pyridine, a substituted pyridine, diisopropylethylamine or pentamethylpiperidine when X is CF3SO3. The most preferable one is 40-O-(2-hydroxy)ethyl rapamycin, which is disclosed in WO94/09010, the disclosure of which is incorporated herein by reference.
The tricyclic compounds (III), and rapamycin and its derivatives, may be in a form of its salt, which includes conventional non-toxic and pharmaceutically acceptable salt such as the salt with inorganic or organic bases, specifically, an alkali metal salt such as sodium salt and potassium salt, an alkali earth metal salt such as calcium salt and magnesium salt, an ammonium salt and an amine salt such as triethylamine salt and N-benzyl-N-methylamine salt.
With respect to the IL-2 inhibitor of the present invention, particularly the tricyclic macrolide compounds, it is to be understood that there may be conformers and one or more stereoisomers such as optical and geometrical isomers due to asymmetric carbon atom(s) or double bond(s), and such conformers and isomers are also included within the scope of the present invention. And further, the tricyclic macrolide compounds can be in the form of a solvate, which is included within the scope of the present invention. The solvate preferably include a hydrate and an ethanolate.
Further example of the IL-2 inhibitor is cyclosporin and its derivatives such as cyclosporin A, B, C, D, E, F, G, etc, which are shown in THE MERCK INDEX (12thedition), No. 2821, U.S. Pat. Nos. 4,117,118, 4,215,199, 4,288,431, 4,388,307, Helv. Chim. Acta. 60, 1568(1977) and 65, 1655(1982), Transplant. Proc. 17, 1362(1985), and so on. Among which, the most preferable one is cyclosporin A. The disclosures of the above references are incorporated herein.
The tricyclic compounds (III) and its pharmaceutically acceptable salts, and cyclosporin or its derivatives may be classified as xe2x80x9cIL-2 production inhibitorxe2x80x9d, which show immunosuppressive activity by inhibiting the production of IL-2. And rapamycin or its derivatives may be classified as xe2x80x9cIL-2 signal transmission inhibitorxe2x80x9d, which show immunosuppressive activity by inhibiting the transmission of IL-2 signal.
For therapeutic administration, the compound possessing the inhibitory activity on the production of nitric oxide in the present invention is used in the form of a conventional pharmaceutical preparation in admixture with a conventional pharmaceutically acceptable carrier such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral or external administration. The pharmaceutical preparation may be compounded in a solid form such as granule, capsule, tablet, dragee, suppository or ointment, or in a liquid form such as solution, suspension or emulsion for injection, intravenous drip, ingestion, eye drop, etc. If needed, there may be included in the above preparation auxiliary substance such as stabilizing agent, wetting or emulsifying agent, buffer or any other commonly used additives.
The xe2x80x9ccompound possessing the inhibitory activity on the production of nitric oxidexe2x80x9d as the effective ingredient may usually be administered in a amount which can inhibit the production of nitric oxide. In particular, it may be a unit dose of 0.001 mg/kg to 500 mg/kg, preferably 0.01 mg/kg to 10 mg/kg, 1 to 4 times a day. However, the above dosage may be increased or decreased according to age, body weight and conditions of the patient or administering method.
If advisable, the compound possessing an inhibitory activity on the production of nitric oxide can be mixed with the IL-2 inhibitor prior to its use. So, the composition comprising the said compound possessing the inhibitory activity on the production of nitric oxide of the present invention may further comprise the IL-2 inhibitor. And optionally, it comprises further additional ingredients, such as, mycophenolatemofetil (CellCept), steroids, Azathiopurine, and so on.
The following Preparations and Examples are given for the purpose of illustrating the present invention in detail.
In the following Examples and Preparations, there are employed the other abbreviations in addition to the abbreviations adopted by the IUPAC-IUB (Commission on Biological Nomenclature).
The abbreviations used are as follows.
Boc: tert-butoxycarbonyl
Me: methyl
Et: ethyl
Pr: propyl
i-Pr: isopropyl
Bu: butyl
Ph: phenyl
Ts: p-toluenesulfonyl
Ac: acetyl
Bn: benzyl
Cbz: benzlyoxycarbonyl
Tf: trifluoromethanesulfonyl
The starting compounds used and the compounds obtained in the following Preparations are given in the Tables as below, in which the formula of the starting compounds are in the left and the formula of the object compounds are in the right, respectively.
A solution of the starting compound (669 mg) and 40% methylamine(0.7 ml) in a mixture of acetic acid (0.7 ml) and xylene (7 ml) was refluxed for 4 hours ina flask equipped with a Dean-Stark trap. The mixture was concentrated, neutralized with 1N sodium hydroxide solution, and extracted three times with chloroform. The organic layer was washed with brine, dried over magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, chloroform/methanol=50/1) to give the object compound as an oil (445 mg).
MASS (ESI) (m/z): 288 (M+H)+; 1H-NMR (CDCl3, 300 MHz) xcex4: 1.46(9H, s), 3.60(3H, s), 4.48(2H, d, J=5 Hz), 5.33(1H, br s), 6.99(1H, s), 7.30-7.52(5H, m).
To a solution of the starting compound (3.10 g) in methanol (15 ml) was added concentrated hydrochloric acid (3 ml), and the mixture was heated to 50xc2x0 C. for 2 hours. The mixture was concentrated, made basic with a 1N sodium hydroxide solution, and extracted three times with chloroform. The organic layer was dried over magnesium sulfate, and filtered. Evaporation of the solvent gave the object compound(2.35 g).
MASS (ESI) (m/z): 308 (M+H)+; 1H-NMR (CDCl3, 300 MHz) xcex4: 3.02-3.22(2H, m), 3.21(3H, s), 3.78(3H, s), 4.11(1H, t, J=7 Hz), 6.81(2H, d, J=8 Hz), 6.99(2H, d, J=8 Hz), 7.04(1H, s), 7.21-7.48(5H, m).
To an ice-cooled mixture of the starting compound (599 mg), 2-aminoacetophenone hydrochloride (362 mg) and 1-hydroxy-benzotriazole (270 mg) in dichloromethane (6 ml) was added 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (349 mg). The mixture was stirred at room temperature for 12 hours. A saturated aqueous sodium hydrogencarbonate solution was added to the mixture, and then the mixture was extracted three times with chloroform. The organic layer was washed with brine, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by column chromatography (silica gel, chloroform/methanol=70/1) to give the object compound (823 mg).
MASS (ESI) (m/z): 417 (M+H)+; 1H-NMR (CDCl3, 300 MHz) xcex4: 1.41(9H, s), 2.96-3.20(2H, m), 4.47(1H, m), 4.70(2H, AB of ABX, JAB=15 Hz), 5.01(1H, br s), 6.92(1H, br s), 7.13(2H, d, J=8 Hz), 7.24(2H, d, J=8 Hz), 7.41-7.68(3H, m), 7.88-8.00(2H, m).
The object compound was obtained according to a similar manner to that of Preparation 3.
oil; MASS: 450 (M+1); 1H-NMR (CDCl3) xcex4: 1.42(9H, s), 3.20-3.30(1H, m), 3.31-3.42 (1H, m), 4.62-4.73(1H, m), 4.70(2H, d, J=6 Hz), 6.42(1H, br s), 7.15(1H, t, J=6 Hz), 7.21(1H, d, J=6 Hz), 7.23(1H, s), 7.33(1H, s), 7.50(2H, d, J=8 Hz), 7.60(1H, t, J=8 Hz), 7.97(1H, s), 8.00(1H, br s), 8.08(2H, d, J=8 Hz), 8.57 (1H, d, J=8 Hz).
The object compound was obtained according to a similar manner to that of Preparation 1.
MASS (ESI) (m/z): 473 (M+H)+; 1H-NMR (CDCl3, 300 MHz) xcex4: 0.76(3H, t, J=7 Hz), 1.38(9H, s), 1.40-1.60(2H, m), 3.48-3.80(2H, m), 3.88-4.08(2H, m), 5.40-5.60(2H, m), 7.02-7.65(10H, m), 7.92(1H, s), 8.52 (1H, d, J=5 Hz).
The object compound was obtained according to a similar manner to that of Preparation 2.
MASS (ESI) (m/z): 373 (M+H)+; 1H-NMR (CDCl3, 300 MHz) xcex4: 0.78(3H, t, J=7 Hz), 1.36-1.72 (2H, m) 3.42-3.74 (2H, m), 3.85-4.24 (2H, m), 4.81-5.02 (1H, m), 7.08(1H, s), 7.15-7.72(9H, m), 7.93(1H, s), 8.55 (1H, d, J=5 Hz).
To an ice-cooled solution of the starting compound (100 mg) indole-2-carboxylic acid (50 mg) and 1-hydroxybenzotriazole (41.9 mg) in dichloromethane (10 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (71.4 mg). The mixture was stirred at room temperature for 12 hours. A saturated aqueous sodium hydrogencarbonate solution was added to the mixture, and then the mixture was extracted three times with chloroform. The organic layer was washed with brine,dried over magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, chloroform/methanol=70/1) to give the object compound as white powder (50 mg).
MASS (m/z): 466 (M+1); 1H-NMR (CDCl3) xcex4: 1.43(3H, t, J=7 Hz), 3.48(3H, s), 3.60(2H, m), 4.03(2H, q, J=7 Hz), 5.97(1H, m), 6.91(2H, d, J=8 Hz), 6.94(1H, s), 6.99(1H, s), 7.10-7.12(3H, m), 7.17(2H, d, J=8 Hz), 7.37(1H, d, J=8 Hz), 7.50(1H, t, J=8 Hz), 7.63(1H, d, J=8 Hz), 9.41(1H, s).
The compound was obtained according to a similar manner to that of Preparation 7.
MASS (ESI) (m/z): 516 (M+H)+; 1H-NMR (DMSO-d6, 300 MHz) xcex4: 0.64(3H, t, J=7 Hz), 1.31-1.55(2H, m), 3.41-3.67(2 H, m), 3.90-4.28(2H, m), 5.86-6.00(1H, m), 6.97-7.21(5H, m), 7.27(1H, s), 7.29-7.42(2H, m), 7.53(2H, d, J=8 Hz), 7.55-7.68(2H, m), 7.73(2H, d, J=8 Hz), 7.81(1H, s), 8.32(1H, s), 8.49(1H, d, J=5 Hz), 9.09(1H, br d, J=8 Hz), 10.50(1H, br s).
To a solution of 2-amino-1-(4-morpholin-4-ylphenyl)ethan-1-one dihydrochloride (3.71 g), (2S)-(tert-butoxycarbonylamino)-3-(2-pyridyl)propanoic acid (5.73 g) and diphenylphosphoryl azide (3.48 g) in N,N-dimethylformamide (70 ml) was added dropwise N,N-diisopropylethylamine (4.41 ml) at 0xc2x0 C. and the mixture was stirred for 20 minutes. The mixture was heated to ambient temperature and stirred for 8 hours. The resulting mixture was diluted with ethyl acetate (200 ml) and washed successively with water, a saturated aqueous sodium hydrogencarbonate solution and brine. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The residual solid was triturated with ethyl acetate-diisopropyl ether (1: 2) to give (2S)-(tert-butoxycarbonylamino)-N-[2-(4-morpholin-4-ylphenyl)-2-oxoethyl]-3-(2-pyridyl)-propanamide (2.06 g) as off-white crystals.
ESI-NS; 469 (M+H); 1H-NMR (300 MHz, CDCl3) xcex4 1.46(9H, s), 3.20-3.38(6H, m), 3.82-3.88(4H, m), 4.60(2H, d, J=5 Hz), 4.64-4.74 (1H, br), 6.37-6.45(1H, br), 6.86(2H, d, J=9 Hz), 7.14(1H, dd, J=5,8 Hz), 7.21 (1H, d, J=8 Hz), 7.59(1H, t, J=8 Hz), 7.82-7.90(3H, m), 8.56 (1H, d, J=5 Hz).
To a solution of (2S)-(tert-butoxycarbonylamino)-N-[2-(4-morpholin-4-ylphenyl)-2-oxoethyl]-3-(2-pyridyl)-propanamide (2.0 g) in acetic acid (4.0 ml) and xylene (60 ml) was added methylamine (40% in water, 4.0 ml) and the mixture was refluxed for 3 hours in a round-bottomed flask equipped with a Dean-Stark apparatus. The mixture was cooled to ambient temperature and a mixture of acetic acid (4.0 ml) and methylamine (40% in water, 4.0 ml) was added to the solution. The solution was refluxed for 2 hours and cooled to ambient temperature. The solution was extracted with 1N hydrochloric acid (100 ml) and the aqueous layer was washed with ethyl acetate (50 ml). The aqueous layer was made basic with a saturated aqueous sodium hydrogencarbonate solution and extracted with ethyl acetate (100 ml). The organic layer was washed successively with an aqueous sodium hydrogencarbonate solution and brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent; 2% methanol in chloroform) to give (1S)-(tert-butoxy)-N-[1-[1-methyl-5-(4-morpholin-4-ylphenyl)imidazol-2-yl]-2-(2-pyridyl)ethyl]formamide (1.45 g) as yellow crystals.
ESI-MS; 464 (M+H); 1H-NMR (300 MHz, CDCl3) xcex4 1.37(9H, s), 3.17-3.23(4H, m), 3.40(3H, s), 3.41-3.47(2H, m), 3.83-3.92(4H, m), 5.33-5.47(2H, m), 6.93(1H, s), 6.94(2H, d, J=9 Hz), 7.08-7.16(2H, m), 7.21 (2H, d, J=9 Hz), 7.56(1H, t, J=8 Hz), 8.55(1H, d, J=5 Hz).
To a solution of (1S)-(tert-butoxy)-N-[1-[1-methyl-5-(4-morpholin-4-ylphenyl)imidazol-2-yl]-2-(2-pyridyl)ethyl]formamide (1.43 g) in dichloromethane (25 ml) was added trifluoroacetic acid (5.0 ml) at 0xc2x0 C. and the mixture was stirred at ambient temperature for 2.5 hours. The resulting mixture was concentrated in vacuo and the residue was dissolved in water (20 ml). The aqueous layer was made basic with a saturated aqueous sodium hydrogencarbonate solution and extracted with chloroform (80 ml). The organic layer was dried over magnesium sulfate and concentrated in vacuo. The residue was triturated with ethyl acetate-diisopropyl ether (1:2) to give (1S)-[1-methyl-5-(4-morpholin-4-ylphenyl)imidazol-2-yl]-2-(2-pyridyl)-ethylamine (1.02 g) as yellow crystals.
ESI-MS; 364 (M+H); 1H-NMR (300 MHz, CDCl3) xcex4 3.17-3.23(4H, m), 3.27-3.47(2H, m), 3.49(3H, s), 3.84-3.91 (4H, m), 4.58(1H, dd, J=5,8 Hz), 6.95(2H, d, J=9 Hz), 6.97(1H, s), 7.11-7.18(2H, m), 7.23(2H, d, J=9 Hz), 7.59(1H, t, J=8 Hz), 8.58(1H, d, J=5 Hz).
To a solution of (1S)-[1-methyl-5-(4-morpholin-4-ylphenyl)imidazol-2-yl]-2-(2-pyridyl)ethylamine (120 mg), 5-chlorobenzo[d]furan-2-carboxylic acid (68.1 mg) and 1-hydroxybenzotriazole (49.1 mg) in N,N-dimethylformamide (2.0 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (69.6 mg). The mixture was stirred at ambient temperature for 2 hours and allowed to stand overnight. The resulting mixture was diluted with water (20 ml) and extracted with ethyl acetate (25 ml). The organic layer was extracted with 1N hydrochloric acid (15 ml) and the aqueous layer was made basic with a saturated aqueous sodium hydrogencarbonate solution, then extracted with ethyl acetate (20 ml). The organic layer was washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residual solid was treated with hot acetonitrile (1.5 ml) and the mixture was cooled to ambient temperature. The solid was collected by filtration and washed with acetonitrile to give (1S)-(5-chlorobenzo[d]furan-2-yl)-N-[1-[1-methyl-5-(4-morpholin-4-ylphenyl)imidazol-2-yl]-2-(2-pyridyl)ethyl]formamide (89 mg) as off-white crystals.
mp 162-164xc2x0 C.; ESI-MS; 542 (M+H); 1H-NMR (300 MHz, DMSO-d6) xcex4 3.11-3.17(4H, m), 3.42-3.60(2H, m), 3.53(3H, s), 3.70-3.77(4H, m), 5.83(1H, q, J=8 Hz), 6.88(1H, s), 6.99(2H, d, J=9 Hz), 7.18(1H, dd, J=5,8 Hz), 7.26(2H, d, J=9 Hz), 7.30(1H, d, J=8 Hz), 7.47(1H, d, J=8 Hz), 7.59(1H, s), 7.63(1H, d, J=8 Hz), 7.68(1H, d, J=8 Hz), 7.87(1H, s), 8.48(1H, d, J=5 Hz), 9.32(1H, d, J=8 Hz); [xcex1]D=171.90 (CHCl3, c=1.030%).
In order to illustrate the activity of the compounds (I) and (II) the pharmacological test result of the representative compounds (a)xcx9c(g) of the compound (I) and (II), which were obtained in a similar manner to that of the above-mentioned Preparations and/or the above-identified WO 98/27108 (PCT/JP97/04243), are shown in the following.
Test Compounds: 
Test: Assay for Inhibitory Activity on the Production of Nitric Oxide
The murine macrophage cell line RAW264.7 (American Type Culture Collection, No. TIB71) was used in this study. RAW264.7 cells were grown on F75 plastic culture flasks at 37xc2x0 C., 5% in Dulbecco""s modified Eagle""s medium (DMEM) supplemented with L-glutamine, penicillin, streptomycin and 10% heat-inactivated fetal bovine serum. They were removed from culture flasks by rubber cell scraper and were centrifuged and resuspended in DMEM without phenol red. They were plated in 96-well microtiter plates (105 cells per well) and allowed to adhere over 2 hours. The test samples were added and the cells were preincubated for 1 hour. Thereafter the cells were activated with both of lipopolysaccharide (LPS) (1 xcexcg/ml) and interferon xcex3 (INFxcex3) (3 u/ml) for 18-24 hours. An equal volume of Griess reagent (1% sulfanilamide/0.1% N-naphthylethylenediamine dihydrochloride/2.5% H3PO4) was added and the cells were incubated at room temperature for 10 minutes. The absorbance was read at 570 nm using microplate reader and NO2 was measured using NaNO2 as a standard.
Test result